DNA vectors, such as plasmids, are normally circular, extrachromosomal DNA molecules which replicate autonomously within the cells of host organisms. The cells of many unicellular organisms, including some bacteria, contain naturally-occurring wild-type plasmids which contribute various functions to the host cells such as antibiotic resistance and fertility. These wild-type plasmids and derivatives of them are the basic tools of recombinant DNA technology, providing vehicles for the transformation of the cells of host organisms with foreign DNA sequences which code for production, within the transformed cells, of corresponding foreign polypeptide and protein products. Thus, in recombinant DNA techniques, plasmids are cut open at specific sites using restriction enzymes and recombined in vitro with additional DNA sequences including genes coding for desired foreign products, to give recombinant plasmids which may be used to transform appropriate host cells.
These recombinant plasmids, similar to the parent plasmids from which they are derived, are capable of autonomous replication within host cells, and on replication reproduce not only the DNA sequences of the parent plasmid but also the inserted additional DNA sequences, including the foreign genes. During protein synthesis, transcription and translation of the DNA sequences of the recombinant plasmids carried within transformed host cells give rise inter alia to the synthesis of foreign products corresponding to the inserted foreign genes.
One factor which affects the yield of synthesised foreign product is the number of copies of the foreign gene which are present within the transformed cells, i.e. the copy number at which the recombinant plasmid is maintained within the host cells, this being defined normally as the number of copies of the plasmid per host genome. Generally speaking, the higher the copy number of the recombinant plasmid the greater is the yield of foreign product. Both low copy number plasmids, usually maintained within host cells at about 1-10 copies per genome, and high copy number plasmids, usually maintained at from 11 up to several hundred copies per genome, are known. The copy number of a given wild-type replicon is controlled by DNA sequences surrounding and including a DNA sequence which defines the origin of replication. Thus hereinafter we refer to high copy number and low copy number origins of replication.
High copy number plasmids have been used in recombinant systems with a view to obtaining good yields of foreign products. This can lead to undesirable results, however, since many such high copy number plasmids tend not to be maintained stably within transformed cells and may be lost from the cells before they can be grown to sufficient levels to permit bulk production of foreign products. For example, the foreign product may inhibit propagation of the transformed cells or the high copy number plasmids themselves may be inherently unstable.
It is known that the copy numbers of some plasmids can be amplified above normal levels by inhibition of protein synthesis; for instance, by addition of protein synthesis inhibitors such as chloramphenicol to the fermentation medium. However, protein synthesis is required for production of most gene products, and therefore the inhibitor must be removed before synthesis of foreign gene products can take place. The removal of inhibitor requires complicated manipulations and is not always possible.
Various other solutions have been proposed to overcome the problem of stable maintenance of high copy number plasmids in host cells. For example, in UK Patent Specification No. 1,557,774 it has been proposed to use mutant plasmids having a temperature-dependent plasmid copy number pattern such that the plasmid shows a controlled constant plasmid copy number when host bacteria carrying the plasmid are cultivated at one temperature, but an altered plasmid copy number pattern, allowing a much higher or totally uncontrolled copy number, when the host bacteria carrying the plasmid are grown at a different temperature. Thus cells may be propagated to desired production size culture at one temperature at which the plasmid replicates at low copy number and at which its gene products do not significantly inhibit cell growth. The temperature may then be altered, greatly increasing the plasmid copy number and also the corresponding production of gene products. The introduction of copy number temperature dependence in such mutant plasmids, however, may introduce a source of instability into the plasmid, and it is likely that these mutant plasmids may be unstable or subject to loss when cells carrying them are propagated over a prolonged period of time.
The replication of plasmids is controlled by nucleotide sequences contained within the overall DNA sequence of the plasmid. These sequences include a sequence defining the origin of replication at which DNA replication is initiated and often also associated sequences which control the initiation of replication at the origin and the copy number at which the plasmid is maintained. For example, certain plasmids, of which ColE1 is a typical example, have plasmid replication systems having a number of features in common. These systems comprise a DNA sequence defining an origin of replication and upstream thereof a DNA sequence coding for transcription, in opposing directions, of two RNA species, RNAII and RNAI. The RNAII species provides and RNA primer which forms a complex at or near the origin from which DNA synthesis is initiated; the RNAI species interferes with the formation of this initiation complex. Transcription of the two RNA species is controlled by separate promoter sequences associated with the DNA sequences which code for their transcription. In addition there is a small polypeptide (the rop protein) which is believed to interact with the promoter for RNAII; this polypeptide is not essential for replication and its role is unclear. The origin of replication, the RNA coding sequences and associated promoters together provide an internally self-regulated system which controls the replication incompatibility and the copy number of these plasmids. Certain other plasmids, exemplified by RI and some Staphyloccocal plasmids, also control replication initiation at the transcriptional level, but by a messenger RNA species whose product provides an initiation factor, probably a polypeptide, which is involved in DNA replication.
It is an object of the present invention to provide new DNA vectors which have controllable copy number patterns and thereby overcome problems associated with stable maintenance of vectors which replicate at high copy number only, which new vectors will not be subject to the potential instability of previously described mutant plasmids which have temperature-dependent copy number patterns, and which, furthermore, will have the advantage that their copy number can be controlled by agents other than temperature, e.g. metabolite concentration.
Accordingly, in a first aspect the invention provides a DNA vector comprising two replication systems; a first origin of replication resulting in a low copy number and stable inheritance of the vector and a second, high copy number origin of replication at which replication is directly controllable as the result of replacement or alteration by DNA manipulation of the natural vector sequence(s) which control replication at said origin.
By means of the invention, when host cells carrying the vector are propagated under a first set of conditions, replication takes place mainly, and preferably exclusively, from the low copy number origin, and when the cells are propagated under a different set of conditions, replication takes place at high copy number at the second origin as well and the production of large amounts of foreign gene products encoded by the vector is induced.
In a second aspect the invention provides a DNA vector containing a replication system comprising an origin of replication and an associated DNA sequence encoding and RNA species which provides a primer or initiation factor (e.g. a polypeptide) which initiates DNA replication by formation of a complex at or near the origin of replication, in which transcription of said RNA species is directly controllable such that, when host cells carrying the vector are propagated under selected conditions, replication takes place at high copy number from the origin and the production of large amounts of foreign gene products encoded by the vector is initiated.
Preferably the second replication system of the first aspect of the invention is a replication system as specified in the second aspect of the invention.
The invention also includes a method for the preparation of a vector according to the first aspect, comprising including in the DNA sequence coding for the second replication system a DNA sequence which permits direct control of replication at the second origin.
Methods for the preparation of vectors according to the first aspect of the invention suitably comprise ligating a first DNA sequence coding for the replication system comprising the first origin with a second DNA sequence coding for the secondary replication system. The DNA sequence which permits direct control of replication at the second origin may be incorporated into the second DNA sequence either before or after ligation with the first DNA sequence. Thus the invention further provides methods for the preparation of vectors according to the second aspect of the invention, comprising including in the second DNA sequence a DNA sequence which permits control of replication at the second origin by controlling transcription of the RNA species.
The invention further includes a process for the production of a polypeptide, protein or other gene product which comprises transforming host cells with a vector according to the first aspect of the invention, in which said vector contains a gene sequence coding for production of said polypeptide, protein or other gene product, propagating said transformed cells under a first set of conditions at which replication takes place at low copy number mainly, and preferably exclusively, from the first origin, and then propagating said transformed cells under a second set of conditions at which replication takes place at high copy number also (or exclusively) from the second origin and the expression of said polypeptide, protein or other gene product is induced.
By means of the process of the invention, transformed cells are propagated to give the large scale cultures required for economic production of polypeptide, protein or other products under conditions where the vector replicates at low copy number and the instability problems associated with high copy number vectors are avoided, followed by propagation under different conditions where the vector replicates at high copy number with concomitant high yield of polypeptide, protein or other products.
In particular it has been found, when the expression of the gene product is under the control of a promoter which is regulated by cytoplasmic levels of a repressor, that the increase in vector copy number leads to outstripping of the repressor control and high level expression of the gene product. This is so even in the case when the synthesis of the repressor is autoregulated, e.g. when the promoter/repressor system is that of the tryptophan operon.
The control systems which are used to control the copy number of the vectors of the invention may comprise any of the control systems which are known for controlling replication (and/or expression) in recombinant DNA technology. In particular, the copy number of the controllable origin of replication may be controlled by temperature or one or more metabolites or metabolite analogues. Examples of metabolite-dependent systems which may be used include: tryptophan, lactose, galactose, arabinose or any other metabolite or metabolite analogue, the presence, removal or further metabolism of which can be used to achieve transcription from a given promoter.
The controllable replication system used in the vectors of the invention may be derived from high copy number cloning vectors, such as ColE1-like plasmids, e.g. pAT153, NTP1, CloDF13, RSF1030 or P15A, which have copy number control systems which involve transcription of RNAII or a similar RNA species which provides a primer which initiates DNA replication by formation of a complex at or near the origin or replication. In addition other plasmid replication origins whose replication is controlled by an mRNA species and/or its product(s) may provide the controllable replication systems used in the vectors of the invention. Such replication origins may be obtained from Gram negative bacterial species and are exemplified by R1, R6, R100, RP4, or Gram positive bacterial species, which are exemplified by pUB100, pC194 and certain other Staphylococcal plasmids. Furthermore, bacteriophage origins may be used for secondary controllable origin, e.g. those from .lambda., T3, T4, T7, M13,.phi.X174, SPP1, SPO2 etc.
In an important embodiment of the invention, the controllable replication systems may be prepared from such high copy number cloning vectors by replacement of the natural promoter, which promotes transcription of the RNA species, by a controllable promoter, such as the P.sub.L promoter, P.sub.R promoter, P.sub.re promoter, P'.sub.R promoter, T.sub.7 late promoters, trp promoter, tac promoter, lac promoter, gal promoter, ara promoter or recA promoter (the origin of replication in such a system is termed a"hybrid origin"). Alternatively the natural promoter may be used and transcription of the RNA species made controllable by incorporating a regulating function, such as an operator sequence, e.g. the lac operator or O.sub.L or O.sub.R operators of phage lambda, into the replication systems.
The plasmids pMG9 (containing an Xho I linker DNA sequence inserted as described by K. Tatchell et al, Cell, Vol 27, pages 25-35, November 1981 (Part 2)) provides a convenient starting material for preparation of a controllable replication system based on the ColE1 replication origin. This plasmid has a unique Xho I restriction site, close to the start of the sequence coding for transcription of the RNAII species, which we have found may be used for insertion of operator and controllable promoter sequences, e.g. .lambda.P.sub.L, to give a directly controllable replication system.
The replication system comprising the first origin of replication may be obtained from any suitable low copy number plasmid. For example, a replication system comprising the pSC101 origin may be used, and the plasmid pHSG415 (T. Hashimoto-Gotoh et al, Gene, 16 (1981) pages 227-235) provides a convenient source for such a replication system. It will be appreciated that pHSG415 has a temperature sensitive replication origin, but that this origin may be replaced by its wild-type temperature-stable counterpart from pSC101. pHSG415, however, provided a convenient, temperature sensitive replication origin for use in the examples hereinafter described.
Controllable functions may be incorporated into high copy number replication systems, high copy number and low copy number replication systems may be ligated, and foreign genes may be incorporated into the vectors to produce vectors according to the invention using techniques which are known and understood by workers skilled in the recombinant DNA art. The resultant vectors may then be used to transform suitable host cells using standard procedures to produce foreign polypeptide, protein and other products.
The host cells may comprise eucaryotic cells, including yeast cells e.g. S. cerevisae, or, more usually, bacterial cells, of species such as B. subtilis or, especially, E. coli.
The invention is further described by way of illustration only in the following Examples, Examples 1-7. These Examples relate to the construction of specific dual origin plasmids according to the invention, and to studies of copy number control, heterologous gene expression and stability of these plasmids. It will be appreciated that the invention is not limited to the specific plasmids and methods described.